Evidence against roles for phorbol binding protein Munc13-1, ADAM adaptor Eve-1, or vesicle trafficking phosphoproteins Munc18 or NSF as phospho-state-sensitive modulators of phorbol/PKC-activated Alzheimer APP ectodomain shedding

<p>Abstract</p> <p>Background</p> <p>Shedding of the Alzheimer amyloid precursor protein (APP) ectodomain can be accelerated by phorbol esters, compounds that act via protein kinase C (PKC) or through unconventional phorbol-binding proteins such as Munc13-1. We have previously demonstrated that application of phorbol esters or purified PKC potentiates budding of APP-bearing secretory vesicles at the <it>trans</it>-Golgi network (TGN) and toward the plasma membrane where APP becomes a substrate for enzymes responsible for shedding, known collectively as α-secretase(s). However, molecular identification of the presumptive "phospho-state-sensitive modulators of ectodomain shedding" (PMES) responsible for regulated shedding has been challenging. Here, we examined the effects on APP ectodomain shedding of four phorbol-sensitive proteins involved in regulation of vesicular membrane trafficking of APP: Munc13-1, Munc18, NSF, and Eve-1.</p> <p>Results</p> <p>Overexpression of either phorbol-sensitive wildtype Munc13-1 or phorbol-insensitive Munc13-1 H567K resulted in increased basal APP ectodomain shedding. However, in contrast to the report of Roßner <it>et al </it>(2004), phorbol ester-dependent APP ectodomain shedding from cells overexpressing APP and Munc13-1 wildtype was indistinguishable from that observed following application of phorbol to cells overexpressing APP and Munc13-1 H567K mutant. This pattern of similar effects on basal and stimulated APP shedding was also observed for Munc18 and NSF. Eve-1, an ADAM adaptor protein reported to be essential for PKC-regulated shedding of pro-EGF, was found to play no obvious role in regulated shedding of sAPPα.</p> <p>Conclusion</p> <p>Our results indicate that, in the HEK293 system, Munc13-1, Munc18, NSF, and EVE-1 fail to meet essential criteria for identity as PMES for APP.</p

Cannabigerolic acid, a major biosynthetic precursor molecule in cannabis, exhibits divergent effects on seizures in mouse models of epilepsy

Background and Purpose: Cannabis has been used to treat epilepsy for millennia, with such use validated by regulatory approval of cannabidiol (CBD) for Dravet syndrome. Unregulated artisanal cannabis-based products used to treat children with intractable epilepsies often contain relatively low doses of CBD but are enriched in other phytocannabinoids. This raises the possibility that other cannabis constituents might have anticonvulsant properties. Experimental Approach: We used the Scn1a+/− mouse model of Dravet syndrome to investigate the cannabis plant for phytocannabinoids with anticonvulsant effects against hyperthermia-induced seizures. The most promising, cannabigerolic acid (CBGA), was further examined against spontaneous seizures and survival in Scn1a+/− mice and in electroshock seizure models. Pharmacological effects of CBGA were surveyed across multiple drug targets. Key Results: The initial screen identified three phytocannabinoids with novel anticonvulsant properties: CBGA, cannabidivarinic acid (CBDVA) and cannabigerovarinic acid (CBGVA). CBGA was most potent and potentiated the anticonvulsant effects of clobazam against hyperthermia-induced and spontaneous seizures, and was anticonvulsant in the MES threshold test. However, CBGA was proconvulsant in the 6-Hz threshold test and a high dose increased spontaneous seizure frequency in Scn1a+/− mice. CBGA was found to interact with numerous epilepsy-relevant targets including GPR55, TRPV1 channels and GABAA receptors. Conclusion and Implications: These results suggest that CBGA, CBDVA and CBGVA may contribute to the effects of cannabis-based products in childhood epilepsy. Although these phytocannabinoids have anticonvulsant potential and could be lead compounds for drug development programmes, several liabilities would need to be overcome before CBD is superseded by another in this class

Breast cancer management pathways during the COVID-19 pandemic: outcomes from the UK ‘Alert Level 4’ phase of the B-MaP-C study

Abstract: Background: The B-MaP-C study aimed to determine alterations to breast cancer (BC) management during the peak transmission period of the UK COVID-19 pandemic and the potential impact of these treatment decisions. Methods: This was a national cohort study of patients with early BC undergoing multidisciplinary team (MDT)-guided treatment recommendations during the pandemic, designated ‘standard’ or ‘COVID-altered’, in the preoperative, operative and post-operative setting. Findings: Of 3776 patients (from 64 UK units) in the study, 2246 (59%) had ‘COVID-altered’ management. ‘Bridging’ endocrine therapy was used (n = 951) where theatre capacity was reduced. There was increasing access to COVID-19 low-risk theatres during the study period (59%). In line with national guidance, immediate breast reconstruction was avoided (n = 299). Where adjuvant chemotherapy was omitted (n = 81), the median benefit was only 3% (IQR 2–9%) using ‘NHS Predict’. There was the rapid adoption of new evidence-based hypofractionated radiotherapy (n = 781, from 46 units). Only 14 patients (1%) tested positive for SARS-CoV-2 during their treatment journey. Conclusions: The majority of ‘COVID-altered’ management decisions were largely in line with pre-COVID evidence-based guidelines, implying that breast cancer survival outcomes are unlikely to be negatively impacted by the pandemic. However, in this study, the potential impact of delays to BC presentation or diagnosis remains unknown

Society of Family Planning Clinical Recommendations: Contraceptive Care in the Context of Pandemic Response

The coronavirus disease 2019 (COVID-19) pandemic has posed a burden to healthcare systems around the world and has changed the way people access health services, including contraception. This document sets forth guidance from the Society of Family Planning for providing contraceptive care in the context of the COVID-19 pandemic, including when access to healthcare is restricted due to pandemic response. It also outlines the role of telehealth for providing contraceptive care beyond the pandemic. Clinicians can use synchronous telemedicine visits and other forms of telehealth to provide many aspects of contraceptive care. Both audio-video and audio-only visits are acceptable forms of telemedicine. Access to permanent contraception should be maintained, especially in the postpartum period. Combined hormonal contraceptive (CHC) users who have asymptomatic or mild COVID-19 infection may continue their contraceptive method, while those admitted to the hospital with severe infection should suspend CHC use until they are clinically recovered. CHC users who take Paxlovid for mild-moderate COVID-19 infection can consider a back-up contraceptive method for the duration of therapy, but clinically relevant drug interactions are unlikely. Future research should examine contraceptive outcomes in people who receive care via telemedicine; and access to telemedicine among historically excluded populations such as adolescents, people of color, people of low socioeconomic status, disabled people, or people who do not speak English as a primary language

Authors: Motor Neuron Inhibition-Based Gene Therapy for Spasticity REVIEW &amp; ANALYSIS MECHANISM OF SPASTICITY

ABSTRACT Spasticity is a condition resulting from excess motor neuron excitation, leading to involuntary muscle contraction in response to increased velocity of movement, for which there is currently no cure. Existing symptomatic therapies face a variety of limitations. The extent of relief that can be delivered by ablative techniques such as rhizotomy is limited by the potential for sensory denervation. Pharmacological approaches, including intrathecal baclofen, can be undermined by tolerance. One potential new approach to the treatment of spasticity is the control of neuromuscular overactivity through the delivery of genes capable of inducing synaptic inhibition. A variety of experiments in cell culture and animal models have demonstrated the ability of neural gene transfer to inhibit neuronal activity and suppress transmission. Similarly, enthusiasm for the application of gene therapy to neurodegenerative diseases of motor neurons has led to the development of a variety of strategies for motor neuron gene delivery. In this review, we discuss the limitations of existing spasticity therapies, the feasibility of motor neuron inhibition as a gene-based treatment for spasticity, potential inhibitory transgene candidates, strategies for control of transgene expression, and applicable motor neuron gene targeting strategies. Finally, we discuss future directions and the potential for gene-based motor neuron inhibition in therapeutic clinical trials to serve as an effective treatment modality for spasticity, either in conjunction with or as a replacement for presently available therapies. Key Words: Spasticity, Transgene Expression, Motor Neuron, Gene Targeting Spasti city is a condition resulting from excess motor neuron excitation caused by lesions in the upper motor neuron pathway that lead to the absence of inhibition of alpha and/or gamma motor neurons. This loss of inhibition results in involuntary muscle contraction, causing stiffness interfering with movement, speech, and locomotion. 1,2 Affecting more than 12 million people worldwide, spasticity is commonly caused by stroke, multiple sclerosis, cerebral palsy, cerebral infection (encephalitis/meningitis), and/or cerebrospinal trauma. REVIEW &amp; ANALYSIS Spasticity MECHANISM OF SPASTICITY The exact mechanism of spasticity in humans is incompletely understood, primarily because it is multifactorial in nature. It is generally understood that spasticity is caused by pathology involving the stretch reflex, which normally causes a muscle to contract to resist the force that is stretching it. For normal movement to occur, the brain must be able to selectively turn this reflex off, usually via inhibitory signals relayed to the spinal cord via the corticospinal tract. However, damage to this circuit results in disinhibition of the stretch reflex; over time, this reduces the triggering threshold until excessive and complete muscle contraction can occur even at rest, making the limb virtually impossible to move. Specific causes proposed include (a) alpha motor neuron hyperexcitability resulting from an imbalance in excitatory vs. inhibitory alpha motor neurons, and (b) gamma motor neuron hyperactivity manifesting as increased sensitivity of muscle spindle to stretch (fusimotor hyperactivity). 9 Additional causes involve damage to descending tracts that control interneurons responsible for (a) mediating presynaptic inhibition of the Ia terminals on the alpha motor neuron, (b) mediating type II afferents, and (c) reciprocal Ia inhibition. Such damage results in greater afferent stimulus to the alpha motor neuron as a result of stretch, decreased inhibition from type II afferents, and loss of normal inhibition of antagonist muscle during muscle stretch. Finally, a mechanism of decreased recurrent inhibition from Renshaw cells as a consequence of supraspinal damage has also been proposed. Whereas each individual cause contributes to the clinical picture observed in spasticity, it is unlikely that any single hypothesis is sufficient to explain the exact mechanism of spasticity. EXISTING THERAPIES AND LIMITATIONS Medical Therapies Although there are a number of oral medications available to treat spasticity, almost none are effective without significant side effects. The most common medications are diazepam, baclofen, and progabide, all of which are designed to increase presynaptic inhibition of alpha motor neurons by activation of ␥-aminobutyric acid (GABA) receptors. However, these treatments are associated with a high incidence of adverse effects, including sedation, weakness, fever without infection, and elevated liver enzymes. 10,11 Agents affecting ion flux in skeletal muscle, such as dantrolene, lamotrigine, and riluzole, share side effects of muscle weakness, sedation, and idiosyncratic hepatitis. 11,13 These significant toxicities limit the doses of medication that can be employed, thereby limiting efficacy. Furthermore, the issue of tolerance significantly hinders the longterm efficacy of any pharmacologic therapy, particularly for baclofen and diazepam. An alternative medical therapy involves the use of clostridial toxin (i.e., botulinum toxin), which acts by decreasing acetylcholine release at the neuromuscular junction, resulting in a neuromuscular blocking effect. However, the results from this therapy are often transient, with redosing complicated by tachyphylaxis, and increasing dosage complicated by severe muscle weakness. 14 -16 The issue of cost is another consideration. The cost of clostridial toxin treatment might hinder its extensive clinical applications because conventional oral therapies are much less expensive. 17 Surgical Therapies Surgery for spasticity is reserved for cases refractory to medical management or for those that cannot be medically managed because of intolerable side effects. The most common surgical options are generally orthopedic (consisting primarily of tendon-release operations) and neurosurgical. 18,19 Neurosurgical procedures fall into two categories: nonablative and ablative. The most frequently used nonablative procedure is intrathecal baclofen (ITB), which is generally offered for refractory patients with chronic spasticity (Ͼ12 mos). To be considered a candidate, a patient must demonstrate a positive response to ITB at a test dose of less than 100 g, compared with no response to placebo. 20 Although highly effective in improving muscle tone and reducing postoperative spasticity, ITB is fraught with catheter-and wound-related morbidity, both at the time of implantation and throughout the life of the implanted device. 20,21 There are limited data characterizing the problem of tolerance to ITB. [22][23][24][25] In many cases, ITB tolerance is ascribed to progression of the underlying disease (in amyotrophic lateral sclerosis and multiple sclerosis) or to dynamic catheter obstruction (kinking), which is difficult to demonstrate on standard pump contrast injections (pumpograms) or nuclear medicine studies. 23,26 In our practice, we use inpatient externalized catheter ITB trials to address the question of baclofen tolerance. In this context, it is easy to assess the patency of the catheter and document the threshold for response to an intrathecal drug. Outpatient trials of bolus intrathecal injection can be attempted, but these are often misleading because of the inherent differences in the pharmacokinetics of bolus and pump injection. However, the majority of our patients require gradually escalated doses of ITB to maintain adequate control of spasticity. May 2007 Gene Therapy for Spasticity 413 Another nonablative option is spinal cord stimulation, which has been shown to facilitate spasticity control, spasm inhibition, and gait improvement in spastic patients, 27-29 likely by selective modification of segmental spinal reflexes. 33 The most common ablative procedure is selective dorsal rhizotomy, which uses intraoperative electromyography and stimulation to identify the rootlets most responsible for causing severe spasticity. 42-45 POSSIBLE FUTURE THERAPIES Despite the wide range of medical and surgical treatments for spasticity, there is currently no treatment modality that is hardware free, reversible, adjustable, nondestructive, and not subject to tolerance. 2,3,14 -16,46 A potential modality for satisfying these criteria is the use of viral vectors to elicit effects on muscle contraction via gene transfer, because selective control of certain genes has been shown to modulate neuronal activity in multiple applications. POTENTIAL INHIBITORY TRANSGENES One of the most widely studied inhibitory transgenes is the gene encoding glutamate decarboxylase (GAD), the rate-limiting enzyme required to produce the inhibitory neurotransmitter GABA. In vitro and in vivo studies using retroviral vectors and adeno-associated virus (AAV) vectors have suggested that it is feasible to achieve long-term GAD expression in the CNS. 57 GAD was expressed, and the expression of GAD induced production of GABA in these neurons. These findings suggest that AAV-mediated GAD gene transfer might provide a treatment option for overactive diseases such as Parkinson disease. 57,58 These results have led to an ongoing Phase I trial of GAD gene transfer to the human subthalamic nucleus for medically refractory Parkinson disease patients. Tetanus Toxin Light Chain Our laboratory has focused on the gene for the light chain (LC) fragment of clostridial neurotoxin. The expression of this gene in neurons provides inhibition of synaptic function in transgenic mice via reversible suppression of glutamatergic neurotransmission. 59,60 Clostridial intoxication in neurons involves the production of inactive single-chain clostridial neurotoxin polypeptides, which are released after bacterial lysis. This release converts the polypeptide from an inactive single-chain molecule to an active di-chain molecule composed of a heavy chain (HC) and an LC fragment linked by a single disulfide bond. HC binds axon terminals and triggers internalization of the toxin. Once inside the neuron, reduction of the disulfide bond releases the active LC fragment. The activated LC cleaves the soluble N-ethylmaleimide-sensitive factor attachment receptor proteins responsible for synaptic vesicle membrane fusion. 59 Such specific, reversible, effective neuronal inhibition makes LC a viable transgene candidate for exploring gene-based treatment of spasticity. Inwardly Rectifying Potassium Channel Kir2.1 Another gene of interest as a feasible modulator of motor neuron activity is Kir2.1, which encodes inwardly rectifying potassium (Kir) channels in the heart and brain. For inwardly rectifying potassium channels, the inward flow of potassium ions at subthreshold is greater than the outward flow of potassium ions for the opposite driving force. This inward rectification results when intracellular magnesium ions and polyamines enter the ion channel pore from the cytoplasmic side but are unable to pass through it to the extracellular solution. The block is more intense at decreased membrane potentials as the larger depolarization facilitates the movement of magnesium ions and polyamines into the pore. In contrast, as membrane potentials approach the resting membrane potential, the decreasing depolarization hinders magnesium ion movement into the pore. When the membrane potential exceeds the resting membrane potential (hyperpolarization), magnesium ions become prevented from entering the channel. Inwardly rectifying potassium channels prevent the membrane potential from depolarizing by increasing the membrane potassium conductance. This increase in potassium permeability counterbalances the excitatory synaptic potentials that drive the initial membrane depolarization, hence inhibiting the formation of the action potential. In this way, the Kir2.1 contributes to stabilizing the resting potential at a sufficiently negative level to prevent enough sodium channel availability for action potential in the CNS and heart. Kir2.1 has been demonstrated to inhibit both evoked and spontaneous activity of neurons in vitro. STRATEGIES FOR CONTROL OF TRANSGENE EXPRESSION Inducible Gene Expression System Gene therapy will only prove beneficial as a treatment modality for spasticity if it provides advantages over existing pharmacologic and lesionbased modalities. For this to be accomplished, transgene expression must be both adjustable and reversible. One way to control viral vector-mediated transgene expression is to use inducible promoter elements. Several inducible promoter systems have been developed for this purpose, such as tetracycline, RU-486, rapamycin responsive systems, and the chimeric drosophila/bombyx ecdysone receptor system

Perceptions of Anal Intercourse Among Heterosexual Women: A Pilot Qualitative Study

Introduction: More than one-third of women in the U.S. have engaged in heterosexual anal intercourse (HAI), but little is known regarding women’s perceptions of HAI and motivations for engaging in this sexual behavior. Aim: This study aimed to explore U.S. women’s motivations for engaging in HAI and to investigate how they navigate HAI in the context of sexual relationships. Methods: Semi-structured interviews were conducted with 20 women, ages 18–50 years old, who had engaged in anal intercourse with a male partner within the past 3 months. The interview guide was developed utilizing a conceptual framework based on the Theory of Planned Behavior. Main Outcome Measure: Thematic content analysis was performed, and salient themes were identified. Results: Salient themes were identified in all key components of the construct, including attitudes toward the behavior, subjective norms, and perceived behavioral control. Women’s intent to engage in HAI was influenced by their attitudes toward HAI and level of control and trust with their partners. Primary motivators were partner and personal pleasure and sexual curiosity and experimentation. Conclusion: The Theory of Planned Behavior construct was well suited to explore factors influencing women’s intent to engage in HAI. Most women perceive negative societal norms toward HAI. Although this does not appear to affect intention to engage in HAI, it does affect disclosure of this sexual activity with friends and healthcare providers. It is important for healthcare providers to provide open, non-judgmental counseling regarding HAI to decrease stigma, enhance communication, and improve sexual health.Benson LS, Gilmore KC, Micks EA, et al. Perceptions of Anal Intercourse Among Heterosexual Women: A Pilot Qualitative Study. Sex Med 2019;7:198–206. Key Words: Heterosexual Anal Intercourse, Penile-Anal Intercourse, Sexual Behavior, Theory of Planned Behavior, Qualitative Method

Evidence against roles for phorbol binding protein Munc13-1, ADAM adaptor Eve-1, or vesicle trafficking phosphoproteins Munc18 or NSF as phospho-state-sensitive modulators of phorbol/PKC-activated Alzheimer APP ectodomain shedding-5

<p><b>Copyright information:</b></p><p>Taken from "Evidence against roles for phorbol binding protein Munc13-1, ADAM adaptor Eve-1, or vesicle trafficking phosphoproteins Munc18 or NSF as phospho-state-sensitive modulators of phorbol/PKC-activated Alzheimer APP ectodomain shedding"</p><p>http://www.molecularneurodegeneration.com/content/2/1/23</p><p>Molecular Neurodegeneration 2007;2():23-23.</p><p>Published online 9 Dec 2007</p><p>PMCID:PMC2211485.</p><p></p>ture incubator. Levels of holoAPP were measured from cell lysates with anti-APP antibody 369. Levels of Munc13-1 wild type and Munc13-1 H567K mutant proteins were measured by anti-GFP antibody. Equal protein loading was verified by measuring the levels of actin protein in all cell lysates

Evidence against roles for phorbol binding protein Munc13-1, ADAM adaptor Eve-1, or vesicle trafficking phosphoproteins Munc18 or NSF as phospho-state-sensitive modulators of phorbol/PKC-activated Alzheimer APP ectodomain shedding-4

<p><b>Copyright information:</b></p><p>Taken from "Evidence against roles for phorbol binding protein Munc13-1, ADAM adaptor Eve-1, or vesicle trafficking phosphoproteins Munc18 or NSF as phospho-state-sensitive modulators of phorbol/PKC-activated Alzheimer APP ectodomain shedding"</p><p>http://www.molecularneurodegeneration.com/content/2/1/23</p><p>Molecular Neurodegeneration 2007;2():23-23.</p><p>Published online 9 Dec 2007</p><p>PMCID:PMC2211485.</p><p></p>esence (+) of PDBu. (B) Quantification of 3 such experiments